Skip Navigation


Rheumatology Advance Access originally published online on February 21, 2007
Rheumatology 2007 46(6):920-926; doi:10.1093/rheumatology/kem014
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
46/6/920    most recent
kem014v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Xu, H.
Right arrow Articles by Sun, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xu, H.
Right arrow Articles by Sun, L.
Related Collections
Right arrow Rheumatoid Arthritis
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Anti-malarial agent artesunate inhibits TNF-{alpha}-induced production of proinflammatory cytokines via inhibition of NF-{kappa}B and PI3 kinase/Akt signal pathway in human rheumatoid arthritis fibroblast-like synoviocytes

H. Xu, Y. He, X. Yang, L. Liang, Z. Zhan, Y. Ye, X. Yang1, F. Lian and L. Sun2

Department of Rheumatology and 1Department of Nephrology, The First Affiliated Hospital, SUN Yat-sen University, Guangzhou, Guangdong, PR China and 2Department of Pathology, School of Medicine, Northwestern University, Chicago, IL, USA

Correspondence to: H. Xu, MD, PhD, Department of Rheumatology, The First Affiliated Hospital, SUN Yat-sen University, NO 58 Zhongshan Road 2, Guangzhou, Guangdong 510080, PR China. E-mail: xuhanshi{at}hotmail.com; xuhanshi{at}mail.sysu.edu.cn


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgement
 References
 
Objectives. Recent studies indicate that the anti-malarial agent artemisinin and its derivatives may exert an anti-inflammatory effect. In this study, we explored the effect of artesunate, an artemisinin derivative, on tumour necrosis factor (TNF)-{alpha}-induced production of interleukins, IL-1ß, IL-6 and IL-8, in human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), and further investigated the signal mechanism by which this compound modulates those cytokines' production.

Methods. RA FLS obtained from patients with active RA were stimulated with TNF-{alpha} and incubated with artesunate, and IL-1ß, IL-6 and IL-8 production was measured by ELISA. DNA-binding activity and nuclear translocation of nuclear factor kappa B (NF-{kappa}B) were measured by a sensitive multi-well colourimetric assay and confocal fluorescence microscopy, respectively. Signal transduction proteins expression was measured by western blot.

Results. Artesunate decreased the secretion of IL-1ß, IL-6 and IL-8 from TNF-{alpha}-stimulated RA FLS in a dose-dependent manner. Artesunate also prevented TNF-{alpha}-induced nuclear NF-{kappa}B translocation, DNA-binding activity and gene transcriptional activity, as well as phosphorylation and degradation of I{kappa}B{alpha}, but phosphorylation of p38 mitogen-activated protein kinase, extracelluar signal-regulated kinase and c-Jun N-terminal kinase were unaffected. The production of IL-1ß, IL-6 and IL-8 induced by TNF-{alpha} was decreased by pyrrolidine dithiocarbamate (PDTC), a chemical inhibitor of NF-{kappa}B. These observations suggest that artesunate inhibits production of IL-1ß, IL-6 and IL-8 through inhibition of NF-{kappa}B signalling pathway. We also showed that artesunate prevented Akt phosphorylation. TNF-{alpha}-induced production of IL-1ß, IL-6 and IL-8 was hampered by treatment with the phosphatidylinositol 3 (PI3) kinase inhibitor LY294002, suggesting that inhibition of Akt activation might inhibit IL-1ß, IL-6 and IL-8 production induced by TNF-{alpha}.

Conclusions. Our results indicate that artesunate exerts an anti-inflammatory effect in RA FLS and provide the evidence that artesunate may have therapeutic potential for RA.

KEY WORDS: Artesunate, Cytokines, Rheumatoid arthritis, Signal transduction, Fibroblast-like synoviocytes


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgement
 References
 
Rheumatoid arthritis (RA) is a common chronic inflammatory disease of joints, characterized by inflammation, synovial hyperplasia and abnormal immune responses. Fibroblast-like synoviocytes (FLS) are key contributors in the pathophysiological process of RA [1]. Inflammatory cytokines have been identified as key players in the pathogenesis of RA [2]. Successful anti-cytokine therapies in RA, particularly anti-TNF-{alpha} and anti-IL-1, have demonstrated critical pathogenetic importance of cytokines [3]. TNF-{alpha} is one of the master inflammatory cytokines that essentially initiate synovial inflammation and joint destruction in RA. As a critical regulator of synovitis, in addition to inducing other cytokines synthesis, TNF-{alpha} also activates a broad array of intracellular signal mechanisms [1].

Artesunate is one of semi-synthetic derivatives of artemisinin isolated from decoctions of traditional Chinese medicine Artemisia annua L (qinghao, sweet wormwood) [4]. During recent years artemisinin and its derivatives have emerged as the most effective and safe drugs for the treatment of severe and chloroquine-resistant malaria [5]. Furthermore, in addition to their anti-malarial properties, artemisinin and its derivatives have been demonstrated to affect a number of other cellular biochemical processes [6–8], such as anti-proliferation, anti-angiogenic effects, induction of cell apoptosis and oxidative stress. More recently, there is increasing evidence to support the notion that these compounds may have potential role on anti-inflammation and immune regulation. For instance, artemisinin has been shown to exert an inhibitory effect on inducible nitric oxide synthase synthesis and transcription factor nuclear factor kappa B (NF-{kappa}B) activation in human astrocytoma T67 cells [9]. 3-(12-beta-artemisininoxy) phenoxyl succinic acid (SM735), a novel artemisinin derivative, was also demonstrated to play a potential immunosuppressive role [10]. However, the anti-inflammatory effects of artemisinin and its derivatives in RA are unknown.

In the present study, we investigated the effect of artesunate, an important artemisinin derivative, on TNF-{alpha}-induced proinflammatory cytokines production in human RA FLS, and further investigated the intracellular signalling mechanism by which artesunate modulates these proinflammatory cytokines production.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgement
 References
 
Reagents and antibodies
TNF-{alpha} was obtained from R&D Systems (Minneapolis, MN, USA). Dulbecco's modified Eagle's medium (DMEM/F12), fetal bovine serum (FBS), antibiotics, Trypsin-EDTA, phosphate-buffered saline (PBS) and other products for cell culture were purchased from Invitrogen (Carlsbad, CA, USA). LY294002 hydrochloride and ß-actin antibody were purchased from Sigma Chemicals (St Louis, MO, USA). I{kappa}B{alpha}, phosphorylated-I{kappa}B{alpha} (Ser32) and NF-{kappa}B-p65 antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). Phospho-p38 mitogen-activated protein kinase (MAPK) (Thr180 and Tyr182) antibody, phospho-c-Jun N-terminal kinase (JNK) antibody, phospho-extracelluar signal-regulated kinase (ERK) antibody, ERK antibody, phospho-Akt antibody and Akt antibody were purchased from Cell Signaling Technologies. Artesunate, kindly provided by Guilin Pharmaceutical Factory (Guilin, Guangxi, PR China), were dissolved in dimethyl sulfoxide (DMSO) (final concentration <0.1% v/v).

Cell culture
Synovial tissues were obtained from patients with active RA (five women, aged 48–69 years), according to the revised criteria of the American College of Rheumatology [11], who were undergoing synovectomy or joint replacement. This study was approved by the Medical Ethics Committee of The First Affiliated Hospital, SUN Yat-sen University, and informed consent was obtained from all patients. Synovial tissues were cut into small pieces and digested with collagenase in DMEM/F12 medium for 2 h at 37°C to isolate synoviocytes. The synoviocytes were grown in DMEM/F12 medium containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified incubator at 37°C under 5% CO2. At confluence, the cells were trypsinized and passaged, and used after 3–5 passages. We characterized that cultured synoviocytes are synovial-fibroblast-like cells.

Detection of production of cytokine
RA FLS were stimulated with TNF-{alpha} (100 pg/ml) for 12 h in the presence or absence of artesunate. For studying the role of Akt and NF-{kappa}B on cytokine secretion, cells were pre-treated with LY294002 (20 µM), an inhibitor of phosphatidylinositol 3-kinase (PI3K), and pyrrolidine dithiocarbamate (PDTC, 300 µM), an inhibitor of NF-{kappa}B activity, for 1 h, respectively. The conditioned media were collected, and secreted IL-1ß, IL-6 and IL-8 were measured by ELISA using a commercial kit (R&D) according to the manufacturer's instructions.

Western blot
For each experiment, a total of 5 x 105 cells were seeded, and at subconfluence (~70%), cells were made quiescent for 24 h in DMEM/F12 medium containing 0.5% FBS, and then treated with various agents. Cells were rinsed twice with ice-cold PBS and added 0.5 ml of the ice-cold lysis buffer [50 mM Tris Cl, pH 7.5, 150 mM NaCl, 100 µg/ml phenylmethylsulphonyl fluoride (PMSF), 0.1% sodium dodecyl sulphate (SDS), 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 10 µg/ml aprotinin, 2 µg/ml leupeptin, 10 mM EDTA], incubated for 20 min on ice, and then were scraped and centrifuged. Protein concentrations were determined by the BCA protein assay (Pierce, Rockford, IL, USA). Equal amounts of protein were solubilized in Laemmli buffer (62.5 mM Tris/HCL pH 6.8, 10% glycerol, 2% SDS, 5% ß-mercaptoethanol and 0.00625% bromophenol blue), boiled for 5 min, and then separated by SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were probed with primary antibodies diluted 1:1000 for I{kappa}B{alpha}, ERK and Akt, and 1:500 for phospho-I{kappa}B{alpha}, phospho-Akt, phospho-p38 mitogen-activated protein kinase (MAPK), phospho-c-Jun N-terminal kinase (JNK) and phospho-extracelluar signal-regulated kinase (ERK) in Tris-buffered saline (TBS)-T containing 5% nonfat milk at 4°C overnight. The membranes were incubated with the appropriate secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, Piscataway, NJ, USA) reaction. Each blot is a representative of at least three similar independent experiments.

Confocal laser scanning fluorescence microscopy
RA FLS were grown on glass coverslips. The cells were fixed with aceton and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. The cells were incubated with anti-NF-{kappa}B p65 antibody (diluted 1:100) for 1 h at room temperature, and then incubated with FITC-conjugated secondary antibody (Santa Cruz). After washing in PBS, cells were incubated for 3 min with 0.25 mg/ml 4'-6-diamidono-2-phenylindole, dihydrochloride (DAPI). The coverslips were mounted on glass slides with anti-fade mounting media and examined using a confocal fluorescence microscopy (Zeiss LSM510).

Measurement of NF-{kappa}B DNA-binding activity
DNA-binding activity of NF-{kappa}B was measured by a sensitive multi-well colourimetric assay [12] using a TransAM NF-kappaB Kit (Active Motif, Carlsbad, CA, USA). Briefly, cultured FLS on culture plates were scraped and centrifuged for 10 min at 1500 rpm at 4°C in a HFA 15.2 rotor (Heraeus Instruments, Hanau, Germany). The pellet was resuspended in 100 µl of lysis buffer, and the lysate was centrifuged for 20 min at 15 000 rpm at 4°C in a HFA 15.2 rotor (Heraeus Instruments). (revised portions are underlined). Supernatant constitutes the total protein extract. Cell extracts (5 µg) from each sample were incubated in 96-well plates coated with NF-{kappa}B consensus double-stranded oligonucleotide sequence (5'-AGTTGAGGGGACTTTCCCAGGC-3') (Active Motif ) for 1 h and then with supplied primary NF-{kappa}B antibody (1:500) for 1 h, and subsequently with secondary peroxidase-conjugated antibody (1:1000) for 1 h at room temperature. After a colourimetric reaction, the optical density was read at 450 nm. For competition assays, the cell extracts were incubated with the 22-bp double-stranded DNA, either wild type (see previously) or mutated: 5'-AGTTGAGCTCACTTTCCCAGGC-3' (underline denotes the substitution).

NF-{kappa}B reporter assay
RA FLS were transiently transfected with 1 µg of pNF-{kappa}B-Luc plasmid by using Lipofectamine reagent (Invitrogen) according to the manufacturer's protocol. Twenty-four hours later, transfected cells were starved overnight in serum-free medium and then seeded in 96-well plates. Cells were then treated with different agents, harvested in reporter lysis buffer (Promega). After normalization of transfection efficiency by ß-galactosidase expression, luciferase enzyme activity was then quantified using a reporter assay kit (Clontech, Palo Alto, CA, USA).

MTT test for measurement of cell activity
RA FLS were pre-treated for 24 h with artesunate at different concentrations (ranging from 0.5 to 40 µM), and then incubated with TNF-{alpha} (100 pg/ml) for 12 h. The culture supernatants were removed and the adherent cells were incubated for 30 min at 37°C with a solution of the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium (MTT) salt (1 mg/ml in PBS). The dark blue crystals of formazan produced were dissolved in acidified isopropanol, and formazan quantification was performed at a test wavelength of 570 nm and a reference wavelength of 620 nm.

Statistical analyses
Data are expressed as mean ± S.E.M. For comparison of paired data, the Wilcoxon signed rank test was used; for multiple comparisons, Kruskal–Wallis analysis of variance was used. A P-value ≤0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgement
 References
 
Artesunate are not toxic to RA FLS in the culture conditions used
The viability of RA FLS in culture was evaluated by the MTT-test, as described under ‘Material and Methods’. Up to 40 µM, the compound under study did not reduce the cell viability (data not shown), which indicates that the inhibitory effect observed were not due to cytotoxic effects.

Effect of artesunate on TNF-{alpha}-induced proinflammatory cytokines production in RA FLS
IL-1ß, IL-6 and IL-8 are critical proinflammatory cytokines in pathogenesis of RA. To investigate the possible inhibitory effects of artesunate on TNF-{alpha}-induced proinflammatory cytokines production, RA FLS were pretreated with or without increasing concentration of artesunate ranging from 0.5 to 20 µM for 24 h, and then exposed to TNF-{alpha} (100 pg/ml) for 12 h. As shown in Fig. 1, artesunate alone exhibited no effect on synthesis of IL-1ß, IL-6 and IL-8, and reduced the release of IL-1ß, IL-6 and IL-8 in TNF-{alpha}-stimulated RA FLS in a dose-dependent manner, which became apparent at 5 µM for IL-1ß, IL-6 and at 1 µM for IL-8, and was maximum at concentrations of 10 µM and 20 µM. We also investigated the effect of artesunate on production of IL-10, an anti-inflammatory cytokine in RA. As shown in Fig. 1D, artesunate did not inhibit the synthesis of IL-10 in TNF-{alpha}-stimulated RA FLS, which further confirms the non-toxic effect of artesunate on inhibiting proinflammatory cytokines production.


Figure 1
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 1. Effect of artesunate on TNF-{alpha}-induced production of cytokines in RA FLS. After treatment with or without artesunate (ART) for 24 h, RA FLS were stimulated with 100 pg/ml TNF-{alpha} for 12 h. The levels of IL-1ß (A), IL-6 (B), IL-8 (C) and IL-10 (D) in cultured cell supernatants were measured by ELISA. Data are representative of five independent experiments (means ± S.E.M.) from different patients. *P < 0.05 vs FLS not treated with TNF-{alpha}; **P < 0.05 vs treatment with TNF-{alpha} alone.

 
Effect of artesunate on TNF-{alpha}-induced signal transduction pathways in RA FLS
To investigate the mechanism by which artesunate inhibits proinflammatory cytokines production, we examined the effect of artesunate on several signal transduction pathways in TNF-{alpha}-stimulated RA FLS.

First, we examined the respective effect of artesunate on TNF-{alpha}-induced phosphorylation of p38 MAPK, JNK and ERK. As shown in Fig. 2, artesunate alone did not affect the phosphorylation of these kinases. None of three kinases activities induced by TNF-{alpha} was suppressed by artesunate.


Figure 2
View larger version (49K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 2. Effect of artesunate on phosphorylation of p38 MAPK, JNK and ERK induced by TNF-{alpha} in RA FLS. After treatment with or without artesunate (ART, 10 µM) for 24 h, RA FLS were stimulated with TNF-{alpha} (100 pg/ml) for 15 min. Phosphorylation of p38 MAPK, JNK and ERK induced by TNF-{alpha} was analysed using western blot analysis. Data are representative of three different experiments (means ± S.E.M.).

 
Next, We detected the effect of artesunate on phosphorylation of Akt, a downstream effector of PI3K. As shown in Fig. 3, artesunate inhibited TNF-{alpha}-induced phosphorylated Akt protein expression. We proceeded to investigate whether an inhibitor of PI3K/Akt pathway affects TNF-{alpha}-induced proinflammatory cytokines production. As shown in Fig. 4, treatment with LY294002, an inhibitor of PI3K, inhibited production of IL-1ß, IL-6 and IL-8 in TNF-{alpha}-stimulated RA FLS. These results indicate that artesunate might affect TNF-{alpha}-induced production of IL-1ß, IL-6 and IL-8 through, at least in part, the PI3K/Akt pathway in RA FLS.


Figure 3
View larger version (27K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 3. Effect of artesunate on phosphorylation of Akt induced by TNF-{alpha} in RA FLS. After treatment with or without artesunate (ART, 10 µM) for 24 h, RA FLS were stimulated with TNF-{alpha} (100 pg/ml) for 15 min. Phosphorylation of Akt induced by TNF-{alpha} was analysed using western blot analysis. A representative gel from five separate experiments is shown (A). Bars are representative as means ± S.E.M. from five separate experiments (B). *P < 0.01 vs untreated control; **P < 0.05 vs treatment with TNF-{alpha} alone.

 

Figure 4
View larger version (8K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 4. LY294002 or PDTC abrogates the production of IL-1ß, IL-6 and IL-8 induced by TNF-{alpha} in RA FLS. After treatment with or without artesunate (ART, 10 µM) for 24 h, RA FLS were stimulated with 100 pg/ml TNF-{alpha} for 12 h. The LY294002 (20 µM) and PDTC (300 µM) were added, respectively, 1 h before stimulation with TNF-{alpha}. The levels of IL-1ß (A), IL-6 (B) and IL-8 (C) in the cultured medium were measured by ELISA. Data are representative of five independent experiments (means ± S.E.M.) from five different patients. *P < 0.05 vs FLS not treated with TNF-{alpha}; **P < 0.05 vs treatment with TNF-{alpha} alone.

 
Effect of artesunate on TNF-{alpha}-induced activation of NF-{kappa}B in RA FLS
As the NF-{kappa}B is a major regulator of proinflammatory cytokine expression in TNF-{alpha}-stimulated FLS, we focused the effect of artesunate on the NF-{kappa}B-dependent pathway. To investigate the effect of artesunate on TNF-{alpha}-induced NF-{kappa}B activation, the RA FLS were pre-treated with 10 µM artesunate for 24 h and then incubated with TNF-{alpha} (100 pg/ml) for 30 min. For studying the nuclear translocation of NF-{kappa}B, we performed laser scanning confocal immunofluorescent microcopy using antibody of p65, a major subunit of NF-{kappa}B. As shown in Fig. 5A, immunofluorescence staining showed the translocation of p65 into the nucleus of RA FLS with the treatment of TNF-{alpha}; however, it remained in the cytoplasm of those cells without such treatment. Upon treatment with artesunate (10 µM), TNF-{alpha}-induced nuclear translocation of p65 were prevented.


Figure 5
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 5. Effect of artesunate on TNF-{alpha}-induced activation of NF-{kappa}B. (A) After treatment with or without artesunate (ART, 10 µM) for 24 h, RA FLS were stimulated with 100 pg/ml TNF-{alpha} for 30 min. The nuclear translocation of NF-{kappa}B subunit p65 was assessed by confocal fluorescence microscopy using anti-p65 antibody. Representative laser confocal microcopy images showing nuclear translocation and colocalization of p65 (green stain) with nuclei stained with DAPI (red stain) in cells exposed to TNF-{alpha} (100 pg/ml) with or without ART. (B) Cells were pre-treated 24 h with or without ART (10 µM), and then stimulated with TNF-{alpha} (100 pg/ml) for 30 min. The cells exposed to TNF-{alpha} exhibited increased DNA-binding activity of NF-{kappa}B (blue bar), and ART prevented the effect of TNF-{alpha} on DNA binding. Moreover, each DNA-binding capacity of NF-{kappa}B is effectively competed for by the wild-type consensus oligonucleotide (red bar) but not mutated oligonucleotide (yellow bar). Data are presented as mean ± S.E.M. from five separate experiments using five different RA patients. *P < 0.05 vs untreated control; **P < 0.05 vs treatment with TNF-{alpha} alone.

 
NF-{kappa}B DNA-binding activity was measured by a sensitive multi-well colourimetric assay using specific oligonucleotide probe for NF-{kappa}B. As shown in Fig. 5B, RA FLS treated with TNF-{alpha} showed an increase in NF-{kappa}B DNA-binding capacity. Upon treatment with artesunate (10 M), the increase of TNF-{alpha}-induced NF-{kappa}B binding capacity was also decreased.

Degradation and phosphorylation of I{kappa}B play a critical role in translocation of NF-{kappa}B into the nucleus. For studying the potential involvement of I{kappa}B in TNF-{alpha}-induced signaling pathway, we performed western blot analysis using a specific anti-I{kappa}B{alpha} and anti-phosphorylated I{kappa}B{alpha} antibody, respectively. As shown in Fig. 6A–6C, artesunate (10 µM) alone had no effect on the I{kappa}B{alpha} degradation and phosphorylation; however, it prevented TNF-{alpha}-induced degradation and phosphorylation of I{kappa}B{alpha}. These results indicate that artesunate inhibited TNF-{alpha}-induced NF-{kappa}B activation by preventing I{kappa}B{alpha} degradation and phosphorylation in human RA FLS.


Figure 6
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 6. Effect of artesunate on TNF-{alpha}-induced degradation and phosphorylation of I{kappa}B{alpha}. After treatment with or without artesunate (ART, 10 µM) for 24 h, RA FLS were stimulated with TNF-{alpha} (100 pg/ml) for 15 min. Degradation and phosphorylation of I{kappa}B induced by TNF-{alpha} was analysed using western blot analysis. Data are presented as means ± S.E.M. from five separate experiments (B and C). *P < 0.05 vs untreated control; **P < 0.05 vs treatment with TNF-{alpha} alone.

 
In addition, we also demonstrated that PDTC, a specific inhibitor of NF-{kappa}B, reduced markedly the increase of IL-1ß, IL-6 and IL-8 production induced by TNF-{alpha} (Fig. 4), suggesting the modulation of NF-{kappa}B in TNF-{alpha}-induced cytokines secretion by RA FLS.

Effect of artesunate on TNF-{alpha}-induced NF-{kappa}B gene reporter in RA FLS
To investigate the role of TNF-{alpha} and artesunate on NF-{kappa}B gene transcription in RA FLS, we transfected the cells with an NF-{kappa}B-dependent luciferase gene reporter plasmid. As shown in Fig. 7, TNF-{alpha} stimulation caused a significant increase in NF-{kappa}B-dependent transcription of a luciferase reporter gene, and co-treatment of the cells with artesunate (10 µM) reduced TNF-{alpha}-related NF-{kappa}B transcriptional activity; however, artesunate alone had no effect on the NF-{kappa}B gene transcriptional activity.


Figure 7
View larger version (6K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 7. Effect of artesunate on TNF-{alpha}-induced activation of NF-{kappa}B reporter gene. RA FLS, transfected with an NF-{kappa}B-dependent luciferase gene reporter plasmids, were pre-treated 24 h with or without ART (10 µM), and then stimulated with TNF-{alpha} (100 pg/ml) for 6 h. The cells were harvested for detection of luciferase activity (RLU, relative light unit, mean ± S.E.M.). These data shown are from three separate experiments from three different RA patients. *P < 0.05 vs untreated control; **P < 0.05 vs treatment with TNF-{alpha} alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgement
 References
 
The present study in cultured RA FLS shows for the first time that artesunate suppresses TNF-{alpha}-induced production of IL-1ß, IL-6 and IL-8, suggesting that artesunate has an anti-inflammatory effect on RA FLS. Our results also provide evidence that the mechanism of the inhibitory effect of artesunate on the TNF-{alpha}-induced production of IL-1ß, IL-6 and IL-8 is mediated by NF-{kappa}B signal pathway, and that artesunate also affects the PI3K/Akt pathway. Interestingly, artesunate exerted no effect on phosphorylation of p38 MAPK, ERK and JNK, induced by TNF-{alpha} in RA FLS.

Artemisinin and its derivatives are commonly used drugs in the treatment of patients with severe and chloroquine-resistant malaria [5]. Recent studies indicate that these compounds may exert effects of anti-inflammatory and immune regulation. For instance, a recent study showed that in cultured RAW264.7 cells, artemisinin potently suppressed release of TNF-{alpha} and IL-6 induced by CpG-containing oligodeoxy-nucleotides, lipopolysaccharide or heat-killed Escherichia coli [13]. Wenisch C et al. [14] also reported that artesunate therapy reduced excessive plasma IL-6 levels in patients with severe Plasmodium falciparum malaria. In this study, we found that artesunate inhibits production of IL-1ß, IL-6 and IL-8 in dose-dependent manner by TNF-{alpha}-stimulated RA FLS. FLS plays a critical role in the process of cartilage and bone erosion in RA, presumably through the synthesis of inflammatory mediators including IL-1ß, IL-6 and IL-8. Those proinflammatory cytokines exhibited abundant production in RA synovium and high concentration in the synovial and serum of RA, and have been demonstrated to play critical roles in pathogenesis of RA. Therefore, our results indicate that artesunate may be beneficial for treatment of RA via inhibition of proinflammatory cytokines synthesis.

The intracellular signal mechanisms inducing proinflammatory cytokine production by TNF-{alpha} in RA FLS are not completely understood yet. TNF-{alpha} can bind to its surface receptors and activate several signal transduction pathways such as p38 MAPK, ERK and JNK. It has been reported that these kinases play important roles in chronic synovitis in RA [15, 16], and p38 MAPK mediates IL-6 and IL-8 production in TNF-{alpha}-stimulated RA FLS [17]. However, our results showed that artesunate did not affect phosphorylation of p38 MAPK, ERK and JNK induced by TNF-{alpha}, suggesting that none of those kinases-dependent pathways is involved in the inhibitory effect of artesunate.

NF-{kappa}B is a critical signal transcription factor for regulating immune and inflammatory response, especially in TNF-{alpha}-induced inflammation. In resting cell, NF-{kappa}B is sequestered in the cytoplasm by the inhibitors I{kappa}B family, including I{kappa}B{alpha} and I{kappa}Bß. Once activated by a variety of stimuli, such as proinflammatory cytokines, I{kappa}B proteins are phosphorylated by a complex of I{kappa}B kinases (IKK), ubiquitinated, and rapidly degraded by the proteasome, allowing NF-{kappa}B to release from I{kappa}B and translocate to the nucleus and initiate transcription by binding to numerous specific gene promoter elements [18, 19]. There is increasing evidence suggesting that NF-{kappa}B activation participates in the pathogenesis of RA. For instance, it has been demonstrated that NF-{kappa}B activation is significantly higher in RA synovium than that in osteoarthritis synovium [20, 21]. NF-{kappa}B in cultured FLS is activated rapidly after stimulation by TNF-{alpha} and induces production of many cytokines such as IL-6 and IL-1ß [22, 23]. Several animal models of inflammatory arthritis also demonstrate that inhibition of NF-{kappa}B in vivo can suppress the joint inflammation [24–26]. These studies suggest that NF-{kappa}B may be an attractive therapeutic target for RA [27].

In the present study, we demonstrated that in rheumatoid synoviocytes artesunate inhibits TNF-{alpha}-induced nuclear NF-{kappa}B translocation, DNA-binding activity and gene transcriptional activity, as well as degradation and phosphorylation of I{kappa}B{alpha}. We also found that PDTC, an inhibitor of NF-{kappa}B, reduced increases of IL-1ß, IL-6 and IL-8 secretions induced by TNF-{alpha}. It is well-documented that a great deal of immune and inflammatory genes, including IL-1ß, IL-6 and IL-8 are depended on activation of NF-{kappa}B [28], therefore, our results strongly suggest that artesunate inhibits TNF-{alpha}-induced production of IL-1ß, IL-6 and IL-8 by preventing NF-{kappa}B activation. Indeed, these findings are identical to recent studies that artemisinin suppresses NF-{kappa}B-mediated NO synthesis in human astrocytoma T67 cells [9], and the inhibitory effects of dihydroarteannuin, one of artemisinin derivatives, on elevated TNF-{alpha} production may result from the blockade of NF-{kappa}B signalling pathway in peritoneal macrophages and renal tissue from lupus BXSB mice [29].

Akt, a serine–threonine protein kinase, is a downstream effector of PI3K. Activation of PI3K increases the intracellular amount of phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol–3,4,5-triphosphate, which positively induce Akt phosphorylation. It has been demonstrated that PI3K/Akt pathway can be activated by several cytokines such as TNF-{alpha} [30, 31], TGF-ß [32] and IL-17 [33] in RA synoviocytes. In addition to its effect of anti-apoptosis, PI3K/Akt pathway also mediates some proinflammatory cytokines production in fibroblast [32]. In the present study, we examined whether PI3K/Akt signalling pathway is involved in the inhibitory effect of artesunate in RA FLS. Our results showed that artesunate inhibited TNF-{alpha}-induced Akt phosphorylation. We also observed that production of IL-1ß, IL-6 and IL-8 was hampered by an inhibitor of PI3K, LY294002, in TNF-{alpha}-stimulated RA FLS. Thus, our data suggest that artesunate may also modulate TNF-{alpha}-induced proinflammatory cytokine synthesis via, at least in part, the PI3K/Akt pathway. Although inhibition of PI3K/Akt pathway may induce cellular apoptosis, our study demonstrated that artesunate, the same doses as those, had no toxic effect in RA FLS, indicating that the inhibitory effect of artesunate on cytokines production through PI3K/Akt pathway in our experiments does not result from apoptotic pathway.

In summary, we have demonstrated for the first time that artesunate attenuates TNF-{alpha}-induced production of IL-1ß, IL-6 and IL-8 via inhibition of NF-{kappa}B and PI3K-Akt signalling pathway in RA FLS, suggesting that artesunate may be effective in ameliorating synovial inflammation of RA.

Formula


   Acknowledgement
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgement
References
 
This study was supported in part by a grant for scientific and technological project of Guangdong Province, China (NO.2006B36003014).

The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgement
 References
 

  1. Huber LC, Distler O, Tarner I, Gay RE, Gay S, Pap T. Synovial fibroblasts: key players in rheumatoid arthritis. Rheumatology (2006) 45:669–75.[Abstract/Free Full Text]
  2. Feldmann M, Brennan FM, Maini RN. Role of cytokines in rheumatoid arthritis. Annu Rev Immunol (1996) 14:397–402.[CrossRef][ISI][Medline]
  3. Feldmann M. Pathogenesis of arthritis: recent research progression. Nat Immunol (2001) 2:771–3.[CrossRef][ISI][Medline]
  4. Meshnick SR, Taylor TE, Kamchonwongpaisan S. Artemisinin and the antimalarial endoperoxides: from herbal remedy to targeted chemotherapy. Microbiol Rev (1996) 60:301–15.[Abstract/Free Full Text]
  5. Haynes RK. Artemisinin and derivatives: the future for malaria treatment? Curr Opin Infect Dis (2001) 14:719–26.[ISI][Medline]
  6. Wartenberg M, Wolf S, Budde P, et al. The antimalaria agent artemisinin exerts antiangiogenic effects in mouse embryonic stem cell-derived embryoid bodies. Lab Invest (2003) 83:1647–55.[CrossRef][ISI][Medline]
  7. Huan-huan C, Li-li Y, Shang-bin L. Artesunate reduces chicken chorioallantoic membrane neovascularisation and exhibits antiangiogenic and apoptotic activity on human microvascular dermal endothelial cell. Cancer Lett (2004) 211:163–73.[CrossRef][ISI][Medline]
  8. Efferth T. Molecular pharmacology and pharmacogenomics of artemisinin and its derivatives in cancer cells. Curr Drug Targets (2006) 7:407–21.[CrossRef][ISI][Medline]
  9. Aldieri E, Atragene D, Bergandi L, et al. Artemisinin inhibits inducible nitric oxide synthase and nuclear factor NF-{kappa}B activation. FEBS Letters (2003) 552:141–4.[CrossRef][ISI][Medline]
  10. Zhou WL, Wu JM, Wu QL, et al. A novel artemisinin derivative, 3-(12-beta-artemisininoxy) phenoxyl succinic acid (SM735), mediates immunosuppressive effects in vitro and in vivo. Acta Pharmacol Sin (2005) 26:1352–8.[CrossRef][ISI][Medline]
  11. Arnett FC, Edworthy SM, Bloch DA, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum (1988) 31:315–24.[ISI][Medline]
  12. Renard P, Ernest I, Houbion A, et al. Development of a sensitive multi-well colorimetric assay for active NF-kappaB. Nucl Acid Res (2001) 29:E21.[CrossRef][Medline]
  13. Wang J, Zhou H, Zheng J, et al. The antimalarial artemisinin synergizes with antibiotics to protect against Lethal live Escherichia challenge by decreasing proinflammatory cytokine release. Antimicrob Agents Chemother (2006) 50:2420–7.[Abstract/Free Full Text]
  14. Wenisch C, Linnau KF, Looaresuwan S, Rumpold H. Plasma levels of the interleukin-6 cytokine family in persons with severe Plasmodium falciparum malaria. J Infect Dis (1999) 179:747–50.[CrossRef][ISI][Medline]
  15. Schett G, Tohidast-Akrad M, Smolen JS, et al. Activation, differential location, and regulation of the stress-activated protein kinases, extracelluar signal-regulated kinase, c-JUN N-terminal kinase, and p38 mitogen-activated protein kinase, in synovial tissue and cells in the rheumatoid arthritis. Arthritis Rheum (2000) 43:2501–12.[CrossRef][ISI][Medline]
  16. Han Z, Boyle DL, Aupperle KR, Bennett B, Manning AM. Jun N-Terminal kinase in rheumatoid arthritis. J Pharma Exp Ther (1999) 291:124–30.[Abstract/Free Full Text]
  17. Suzuki M, Tetsuka T, Yoshida S, et al. The role of p38 mitogen-activated protein kinase in IL-6 and IL-8 production from the TNF-alpha- or IL-1 beta-stimulated rheumatoid synovial fibroblasts. FEBS Lett (2000) 465:23–7.[CrossRef][ISI][Medline]
  18. Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-{kappa}B activity. Annu Rev Immunol (2000) 18:621–33.[CrossRef][ISI][Medline]
  19. Baldwin AS Jr. The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu Rev Immunol (1996) 14:649–83.[CrossRef][ISI][Medline]
  20. Han ZN, Boyle DL, Manning AM, Firestein GS. AP-1 and NF-{kappa}B regulation in rheumatoid arthritis and murine collagen-induced arthritis. Autoimmunity (1998) 28:197–208.[ISI][Medline]
  21. Handel ML, McMorrow LB, Gravallese EM. Nuclear factor{kappa}B in rheumatoid synovium: localization of p50 and p65. Arthritis Rheum (1995) 38:1762–70.[ISI][Medline]
  22. Granet C, Maslinski W, Miossec P. Increased AP-1 and NF-{kappa}B activation and recruitment with the combination of the proinflammatory cytokines IL-1, tumor necrosis factor alpha and IL-17 in rheumatoid synoviocytes. Arthritis Res Ther (2004) 6:R190–8.[CrossRef][ISI][Medline]
  23. Miyazawa K, Mori A, Yamamoto K, Okudaira H. Constitutive transcription of the human interleukin-6 gene by rheumatoid synoviocytes: spontaneous activation of NF-{kappa}B and CBF1. Am J Pathol (1998) 152:793–803.[Abstract]
  24. Seetharaman R, Mora AL, Nabozny G, Boothby M, Chen J. Essential role of T cell NF-kappaB activation in collagen-induced arthritis. J Immunol (1999) 163:1577–83.[Abstract/Free Full Text]
  25. Miagkov AV, Kovalenko DV, Brown CE, et al. NF-kappaB activation provides the the potential link between inflammation and hyperplasia in the athritic joint. Proc Natl Acad Sci USA (1998) 95:13859–64.[Abstract/Free Full Text]
  26. Tak PP, Gerlag DM, Aupperle KR, et al. Inhibitor of nuclear factor {kappa}B is a key regulator of synovial inflammation. Arthritis Rheum (2001) 44:1897–907.[CrossRef][ISI][Medline]
  27. Hammaker D, Sweeney S, Firestein GS. Signal transduction networks in rheumatoid arthritis. Ann Rheum Dis (2003) 62:1186–9.
  28. Chabot-Fletcher M. A role for transcription factor NF-{kappa}B in inflammation. Inflamm Res (1997) 46:1–2.[ISI][Medline]
  29. Li WD, Dong YJ, Tu YY, Lin ZB. Dihydroarteannuin ameliorates lupus symptom of BXSB mice by inhibiting production of TNF-{alpha}and blocking the signaling pathway NF-kappa B translocation. Int Immunopharmacol (2006) 6:1243–50.[CrossRef][ISI][Medline]
  30. Morel J, Audo R, Hahne M, Combe B. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces rheumatoid arthritis synovial fibroblast proliferation through mitogen-activated protein kinases and phosphatidylinositol 3-kinase/Akt. J Biol Chem (2005) 280:15709–18.[Abstract/Free Full Text]
  31. Chen Q, Casali B, Pattacini L, Boiardi L, Salvarani C. Tumor necrosis factor-alpha protects synovial cells from nitric oxide induced apoptosis through phosphoinositide 3-kinase Akt signal transduction. J Rheumatol (2006) 33:1061–8.[ISI][Medline]
  32. Kim G, Jun J, Elkon K. Necessary role of phosphatidylinositol 3-kinase in transforming growth factor ß-mediated activation of Akt in normal and rheumatoid arthritis synovial fibroblasts. Arthritis Rheum (2002) 46:1504–11.[CrossRef][ISI][Medline]
  33. Hwang SY, Kim JY, Kim KW, et al. IL-17 induces production of IL-6 and IL-8 in rheumatoid arthritis synovial fibroblast via NF-{kappa}B and PI3-kinase/Akt-dependent pathways. Arthritis Res Ther (2004) 6:R120–8.[CrossRef][ISI][Medline]
Submitted 11 September 2006; revised version accepted 5 January 2007.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
46/6/920    most recent
kem014v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Xu, H.
Right arrow Articles by Sun, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xu, H.
Right arrow Articles by Sun, L.
Related Collections
Right arrow Rheumatoid Arthritis
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?